Summary

Augmented activities of both arginase and S6K1 are involved in endothelial dysfunction in aging. This study was to investigate whether or not there is a crosstalk between arginase and S6K1 in endothelial inflammation and aging in senescent human umbilical vein endothelial cells and in aging mouse models. We show increased arginase-II (Arg-II) expression/activity in senescent endothelial cells. Silencing Arg-II in senescent cells suppresses eNOS-uncoupling, several senescence markers such as senescence-associated-β-galactosidase activity, p53-S15, p21, and expression of vascular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1). Conversely, overexpressing Arg-II in nonsenescent cells promotes eNOS-uncoupling, endothelial senescence, and enhances VCAM1/ICAM1 levels and monocyte adhesion, which are inhibited by co-expressing superoxide dismutase-1. Moreover, overexpressing S6K1 in nonsenescent cells increases, whereas silencing S6K1 in senescent cells decreases Arg-II gene expression/activity through regulation of Arg-II mRNA stability. Furthermore, S6K1 overexpression exerts the same effects as Arg-II on endothelial senescence and inflammation responses, which are prevented by silencing Arg-II, demonstrating a role of Arg-II as the mediator of S6K1-induced endothelial aging. Interestingly, mice that are deficient in Arg-II gene (Arg-II−/−) are not only protected from age-associated increase in Arg-II, VCAM1/ICAM1, aging markers, and eNOS-uncoupling in the aortas but also reveal a decrease in S6K1 activity. Similarly, silencing Arg-II in senescent cells decreases S6K1 activity, demonstrating that Arg-II also stimulates S6K1 in aging. Our study reveals a novel mechanism of mutual positive regulation between S6K1 and Arg-II in endothelial inflammation and aging. Targeting S6K1 and/or Arg-II may decelerate vascular aging and age-associated cardiovascular disease development.

Fig. S4 Silencing Arg-II prevents S6K1-induced Arg-II expression and arginase activity. Young HUVECs were first transduced either with rAd/U6-LacZshRNA as control or rAd/U6-Arg-IIshRNA. Four days post transduction with rAd/U6-shRNA, cells were then transduced either with rAd/CMV as control (con) or rAd/CMV-HA-S6K1ca (a constitutively active S6K1 mutant). Experiments were performed on day two post 2nd transduction. Cells were serum starved with 0.2% FCS-RPMI 12 h prior to experiments. Blots above reveal the immunoblotting analysis of HA-tagged S6K1ca and Arg-II with anti-HA and anti-Arg-II antibody, respectively. Bar graphs below show quantifications of Arg-II/tubulin protein level, arginase activity, and Arg-II/GAPDH mRNA levels as analysed by qRT-PCR. ***p < 0.005 vs. control; †††p < 0.005 vs. S6K1ca.

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